Extraction and Winterization of Lipids from Oilseed and Microbial Sources

ABSTRACT

A process for purifying a lipid composition having predominantly neutral lipid components having at least one long chain polyunsaturated fatty acid is disclosed. The process employs contacting the lipid composition with a polar solvent, such as acetone, wherein the solvent is selected such that contaminants are less soluble in the solvent than is the long chain polyunsaturated fatty acid. The process is typically conducted at cooler temperatures, including about 0° C. Upon precipitation of the contaminants from the lipid composition, a separation is conducted to remove the precipitated material from the lipid composition. The long chain polyunsaturated fatty acids can include ARA, DPA, EPA, and/or DHA. The process of the present invention effectively winterizes lipid compositions, thereby reducing the tendency of such compositions to become hazy.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/498,598, file Dec. 14, 2004, which is a 371 national phaseapplication of PCT Application Ser. No. PCT/US2002/39930, filed Dec. 12,2002, which claims the benefit of priority under 35 U.S.C. § 119(e) ofU.S. Provisional Application Ser. No. 60/341,180, filed Dec. 12, 2001.The disclosure of this application is incorporated by reference hereinin its entirety.

FIELD OF THE INVENTION

The present invention is directed to the extraction and purification oflipids, and in particular, lipids containing long chain polyunsaturatedfatty acids (LCPUFAs). In particular, processes are provided forobtaining high concentrations of desired LCPUFAs and low concentrationsof undesired compounds such as trisaturated glycerides.

BACKGROUND OF THE INVENTION

In general, winterization is the name given to the process of removingsediment that appears in vegetable oils at low temperature. Itoriginated from the early practice of allowing cottonseed oil to remainin outdoor storage during the cool winter months and filtering off thesediment-free oil. Dry fractional crystallization is a process whereintriglycerides with the highest melting temperature preferentiallycrystallize during cooling from a neat liquid (e.g., liquid lipid).After crystallization is complete, the solid phase is separated from theliquid phase by one of several types of physical processes.Alternatively, solvent crystallization is used to promote triglyceridecrystal formation, because triglycerides at low temperature generallyform more stable crystals with solvent than without solvent.

Docosahexaenoic acid (DHA)-rich lipid was extracted using conventionaltechniques and solvents (e.g., hexane) from Schizochytrium sp. biomassproduced by fermentation, and the resulting extracted lipid waswinterized by chilling it to −2 to 2° C. followed by centrifugation. Thelipid was then refined, bleached and deodorized, and put into gelatincapsules for sale as nutritional supplements. A problem arose with thisproduct in that a haze would form in the product over time.

In one process for recovering lipids from biomass, as illustrated inFIG. 1, dried microalgae are suspended in commercial-grade n-hexane andwet milled. Hexane primarily extracts triglycerides, diglycerides,monoglycerides and esterified sterols, although other components of thetotal lipid fraction, such as phospholipids, free sterols andcarotenoids, can also be extracted to a lesser degree. Centrifugation isemployed to separate spent biomass from a lipid-rich miscella. Theresultant mixture of lipid and solvent is referred to as miscella. Thelipid content of the clarified miscella is adjusted to about 45 wt %using n-hexane. The miscella is winterized, in particular, the miscellais chilled to approximately −1° C., and held for 8 to 12 hours, tocrystallize any saturated fats, or high melting point components. Themiscella is then filtered to remove the crystallized stearine phase.Hexane is removed from the miscella, leaving behind the winterizedlipid.

As illustrated in FIG. 2, the winterized lipid is heated and treatedwith citric acid or phosphoric acid to hydrate any phosphatides presentin the lipid. Sodium hydroxide is added to neutralize any free fattyacids present. The resulting gums (hydrated phosphatides) and soapstock(neutralized fatty acids) are removed using a centrifuge. The lipid ismixed with water and re-centrifuged to remove any residualgum/soapstock. This step can be carried out with the firstcentrifugation. The refined lipid is bleached with silica and bleachingclay following pre-treatment with citric acid, to remove peroxides,color compounds, and traces of soapstock, phospholipids and metals.Filter aid is added at the end of the cycle to facilitate removal of thespent bleaching compounds from the lipid via filtration.

An additional step can be performed, where the bleached lipid is chilledto from about 5° C. to about 15° C. and held for about 6 to about 8hours to crystallize any remaining stearines or waxes, if it is apparentthat a sediment layer will form upon standing. Filter aid can be used tofacilitate removal of the crystals via filtration, if this step isperformed.

A deodorizer, operated at elevated temperatures under high vacuum, isused to destroy peroxides, which if left intact could later decomposeand initiate free radical reactions. This step also removes anyremaining low molecular weight compounds that can cause off-odors andflavors. Contact times in the deodorizer are minimized to prevent theformation of trans-fatty acids. Safe and suitable food approvedantioxidants are added. The stabilized lipid is packaged in aphenolic-lined metal container under a nitrogen atmosphere to preventoxidation.

The haze that formed in the lipid-filled gelatin capsules was analyzedand found to be composed of crystals of triglycerides containingmyristic (14:0) and palmitic (16:0) fatty acids, a trisaturated fattyacid glyceride. These crystals had a melting point of about 50-55° C.The trisaturated glycerides comprised 6-8% of the crude extracted lipid.The above-described winterization process lowered the concentration ofthese trisaturated glycerides to <1%; however, not low enough tocompletely eliminate haze formation in the lipid. Additionally, about30% of the lipids, and a corresponding 30% of the DHA, is removed inthis traditional hexane (55% hexane and 45% crude oil) winterizationprocess. Another problem was that when the temperature was lowered tocrystallize the remaining <1% of the trisaturated triglycerides, more ofthe desired LCPUFA, e.g., disaturated triglycerides containing one DHAmolecule, would also crystallize out. This would cause significantlosses of the target product, DHA. Losses could be an additional 8-10%of the lipids,. So by trying to solve one problem, another was created.It would be desirable to have a process by which the LCPUFA level couldbe maintained at a desirably high level and the haze could be reduced oreliminated.

SUMMARY

The present invention includes a process for purifying a lipidcomposition having predominantly neutral lipid components wherein thecomposition contains at least one long chain polyunsaturated fatty acid(LCPUFA) and at least one other compound. The process includescontacting the lipid composition with a polar solvent and the solvent isselected such that the other compound is less soluble in the solventthan is the LCPUFA. For example, the polar solvent can be selected fromacetone, isopropyl alcohol, methanol, ethanol, ethyl acetate andmixtures thereof. The process further includes maintaining the lipidcomposition at a temperature range effective to precipitate at least aportion of the other compound. For example, the temperature range can befrom about −20° C. to about 50° C., from about −5° C. to about 20° C.,from about −5° C. to about 5° C. or about 0° C. The process thenincludes removing at least a portion of the other compound from thelipid composition to form a lipid product. The process can bespecifically for the reduction of the formation of haze in a lipidcomposition in which the compound being removed is a haze-formingcompound.

In various embodiments, the lipid composition can include at least 50%or 85% neutral lipid, or at least 50% triglyceride. The concentration ofLCPUFA, on a weight percentage basis, can be greater after the processthan before, and the concentration of the other compound, on a weightpercentage basis, can be less after the process than before. Forexample, the total concentration of any phosphorus-containing compoundspresent in the lipid, on a weight percentage basis, is less after theprocess than before. The process of the present invention can result inan acceptable product with less downstream processing required, such aswith reduced degumming or no degumming required.

The LCPUFA can be arachidonic acid (ARA), omega-6 docosapentaenoic acid(DPA(n-6)), omega-3 docosapentaenoic acid (DPA(n-3)), eicosapentaenoicacid (EPA) and/or docosahexaenoic acid (DHA). The other compound can betrisaturated glycerides, phosphorus-containing materials, wax esters,saturated fatty acid containing sterol esters, sterols, squalene, and/orhydrocarbons. Alternatively, the other compound can be trisaturatedglycerides, phosphatides and wax esters. Alternatively, the othercompound can be trisaturated glycerides of lauric (C12:0), myristic(C14:0), palmitic (C16:0) and stearic (C18:0) fatty acids and/ormixtures thereof. In a particular embodiment, the lipid compositioninitially comprises at least one LCPUFA and at least one trisaturatedglyceride. The LCPUFA can be obtained from a LCPUFA-containingbiomaterial selected from LCPUFA-containing microbial biomass andoilseeds from plants that have been genetically modified to produceLCPUFA-containing lipid. Also, the LCPUFA can be obtained from plantsthat have been modified with LCPUFA-producing genes from microbes. Inanother embodiment, the LCPUFA can be obtained from a source selectedfrom the group consisting of thraustochytrid biomass, dinoflagellatebiomass, Mortierella biomass, and oilseeds from genetically modifiedplants containing genes from thraustochytrids, dinoflagellates orMortierella. In a further embodiment, the LCPUFA is obtained from thegroup comprising Schizochytrium, Thraustochytrium or Crypthecodiniumcohnii biomass or oilseeds from genetically modified plants containinggenes from Schizochytrium or Thraustochytrium.

In various embodiments of the invention, the solvent:lipid compositionratio is from about 1:10 to about 20:1, from about 1:8 to about 10:1,from about 1:5 to about 5:1, from about 1:2 to about 2.5:1, or about1:1. In other embodiments, the time of contact between the solvent andthe lipid composition is from about 0.5 to about 12 hours, from about 2to about 6 hours, or about 4 hours.

In another embodiment of the invention, lipid is extracted using thepolar solvent at low temperatures such that triglyceride moleculescontaining the LCPUFA are selectively extracted and other compounds thatare not soluble in the polar solvent are not extracted. In a furtherembodiment, the lipid composition is extracted from a biomass andcellular debris and precipitated other compounds are separated from amiscella comprising the LCPUFA and the polar solvent.

A further embodiment of the invention includes employing the polarsolvent to recover lipid in an extraction process conducted attemperatures that solubilize substantially all triglyceride components;forming a miscella comprising a mixture of the lipid composition and thepolar solvent; cooling the miscella to selectively precipitate theundesired compounds; and separating the precipitated other compoundsfrom the miscella. In this embodiment, the lipid composition can beextracted from biomass and cellular debris and precipitated othercompounds are separated from a miscella comprising the LCPUFA and thepolar solvent.

Another embodiment of the invention includes employing the polar solventto recover lipid from a biomass in an extraction process conducted attemperatures that solubilize substantially all triglyceride components,forming a miscella comprising a mixture of the lipid composition, thepolar solvent and cellular debris. The process further includesseparating the cellular debris from the miscella and cooling themiscella to selectively precipitate the undesired compounds. Finally,the precipitated other compounds are separated from the miscella.

A further embodiment of the invention includes employing a nonpolarsolvent to recover lipid in an extraction process conducted attemperatures that solubilize substantially all triglyceride components,forming a miscella comprising a mixture of the lipid composition and thenonpolar solvent. The process further includes removing most of thenonpolar solvent from the miscella, adding a polar solvent to themiscella, and cooling the miscella to selectively precipitate theundesired compounds. Finally, the precipitated other compounds areseparated from the miscella. A still further embodiment of the inventionincludes employing a nonpolar solvent to recover lipid in an extractionprocess conducted at temperatures that solubilize substantially alltriglyceride components, forming a miscella comprising a mixture of thelipid composition and the nonpolar solvent and winterizing the miscella.Most of the nonpolar solvent is removed from the miscella, and a polarsolvent is added to it. The miscella is cooled to selectivelyprecipitate the undesired compounds which are separated from themiscella. When the nonpolar solvent is removed from the miscella, theresidual nonpolar solvent after removal is from about 0 to about 4weight percent or from about 1 to about 4 weight percent.

In the various embodiments of the invention using a nonpolar solvent,the nonpolar solvent can be hexane. In various embodiments of theinvention employing a separating or removing step for the precipitatedother compound, the step can be a liquid/solid separation technique,such as centrifugation, filtering or combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow diagram of a prior extraction process.

FIG. 2 is a flow diagram of a prior refining, bleaching and deodorizingprocess.

FIG. 3 is a flow diagram of a DHA-rich lipid extraction process of thepresent invention using acetone in one step.

FIG. 4 is a flow diagram of a DHA-rich lipid extraction process of thepresent invention using acetone in two steps.

FIG. 5 is a flow diagram of a DHA-rich lipid hexane extraction processand acetone winterization process of the present invention.

DESCRIPTION OF THE INVENTION

In accordance with the present invention, processes are provided forpreferentially reducing the level of undesired components in a lipid,while maintaining high levels of desired LCPUFAs. As used herein,LCPUFAs are fatty acids with 20 or more carbon atoms and two (preferablythree) or more double bonds. The LCPUFAs can be in a variety of forms,such as phospholipids, free fatty acids and esters of fatty acids,including triglycerides of fatty acids. It will be appreciated that whenreferring to the desired LCPUFA, what is meant is the LCPUFA in the formthat exists in the lipid, most typically a triglyceride, and to a lesserextent mono- and diglycerides. Preferably, the concentration of thedesired LCPUFA, as measured on a weight percent basis, is higher in theresulting lipid product than it is in the starting lipid composition.The undesired components are preferably trisaturated glycerides, such astrisaturated glycerides of lauric (C12:0), myristic (C14:0), palmitic(C16:0) and stearic (C18:0) fatty acids and mixtures thereof. Examplesof other undesired components, in addition to trisaturated glycerides,include phosphorus-containing compounds (e.g., phosphatides orphospholipids), wax esters, saturated fatty acid containing sterolesters, sterols, squalene, hydrocarbons and the like. Preferably, two ormore of the undesired compounds are reduced in the resulting product ascompared to the starting lipid, as measured on a weight percent basis.As used herein, amounts will generally be on a weight percent basis,unless indicated otherwise.

In a preferred embodiment of the present invention the resulting productis subject to less haze or cloudiness when compared to the startinglipid. As a result of the process of the present invention, subsequentprocessing steps such as refining, can be reduced or eliminated. Forexample, subsequent processing steps such as bleaching and/ordeodorizing can help reduce or eliminate the refining (or degumming)step. An example of the refining, bleaching and deodorizing process isset forth in comparative Example 2. If the refining process is noteliminated, it can be reduced by reducing the amount of causticemployed. While not wishing to bound by any theory, it is believed thata primary cause of haze or cloudiness results from trisaturatedtriglycerides. It does not appear to be as important to reduce the mono-and di-substituted triglycerides.

As used herein the term “lipids” will refer generally to a variety oflipids, such as phospholipids; free fatty acids; esters of fatty acids,including triglycerides of fatty acids; sterols; pigments (e.g.,carotenoids and oxycarotenoids) and other lipids, and lipid associatedcompounds such as phytosterols, ergothionine, lipoic acid andantioxidants including beta-carotene, tocotrienols, and tocopherol.Preferred lipids and lipid associated compounds include, but are notlimited to, cholesterol, phytosterols, desmosterol, tocotrienols,tocopherols, ubiquinones, carotenoids and xanthophylls such asbeta-carotene, lutein, lycopene, astaxanthin, zeaxanthin, canthaxanthin,and fatty acids such as conjugated linoleic acids, and omega-3 andomega-6 highly unsaturated fatty acids such as eicosapentaenoic acid,docosapentaenoic acid, and docosahexaenoic acid, arachidonic acid,stearidonic acid, dihomogammalinolenic acid and gamma-linolenic acid ormixtures thereof. For the sake of brevity, unless otherwise stated, theterm “lipid” refers to lipid and/or lipid-associated compounds.

The undesirable components share the common characteristic of beingrelatively insoluble in cold acetone or in an analogous polar solvent.On the other hand, desired LCPUFAs, such as arachidonic acid (ARA),omega-6 docosapentaenoic acid (DPA(n-6)), omega-3 docosapentaenoic acid(DPA(n-3)), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA),are soluble in cold acetone or in an analogous solvent. The keycharacteristic of the solvent, whether it is acetone or an analogouspolar solvent, is that the desirable LCPUFAs are soluble in the solventat the desired temperatures, and the undesirable compounds are notsoluble in the solvent at the same temperatures. A useful guide is toselect solvents that have dielectric constants close to those of acetoneor ethyl acetate. Preferred solvents for use in connection with thepresent invention include acetone and analogous polar solvents such asisopropyl alcohol, methanol, ethanol, ethyl acetate or mixtures of thesesolvents. The solvents are all polar, and the LCPUFAs, with their doublebonds and long carbon chains, are also polar and therefore soluble inthe polar solvents. However, if the solvents are too polar, the LCPUFAsmay not dissolve. The solvent is also preferably useful in foodapplications.

It was unexpectedly found that acetone can be used to selectivelyprecipitate the trisaturated glycerides from the crude lipid. When anunwinterized lot of DHA-rich lipid from Schizochytrium sp. was treatedwith 5 volumes of acetone and chilled, essentially all of thetrisaturated glycerides were removed by crystallization followed bycentrifugation. This process removed little or none of theDHA-containing triglycerides. The resulting winterized lipid contained41% DHA as compared to 37% by the standard winterization process.

There are ways to further utilize this discovery by combining acetone oranalogous solvent extraction with “in-situ” winterization concepts tobetter improve the recovery efficiency of long chain polyunsaturatedfatty acid containing triglycerides at the expense of trisaturatedglycerides or from triglycerides containing two saturated fatty acidsand one mono-unsaturated fatty acid. One advantage of the process of thepresent invention is that less of the desired LCPUFAs are lost. Forexample, in prior processes about 30% of the extracted lipid, whichcontained the desired LCPUFAs, was lost during winterization. Incontrast, the embodiment of the process of the present invention (i.e.,hexane extraction followed by acetone winterization) that is mostdirectly comparable to the prior process results in the loss of onlyabout 7% to about 10% of the starting extracted lipid as a result of theacetone winterization. As a result, in this embodiment of the presentinvention, about 40% or more reduction in yield loss is realized. Thisis a significant improvement over the prior process (hexane extractionand winterization plus full refining, bleaching and deodorizing (RBD)).The largest loss of both DHA and lipid is incurred in the winterizationstep of the prior process.

First, in a preferred process, lipid is extracted using acetone oranalogous polar solvent (instead of hexane) at low temperatures suchthat triglyceride molecules containing LCPUFA are selectively extractedfrom Schizochytrium sp. biomass. A flow diagram of such a process isillustrated in FIG. 3. Due to the selectivity of acetone at lowtemperature (trisaturated glycerides are not soluble in cold acetone,while LCPUFA-containing triglycerides are soluble in cold acetone), itis feasible to selectively remove the LCPUFA-containing triglyceridefrom biomass and thus eliminate the need for a separate winterizationstep. The solvent extraction can be conducted in any suitable manner.For example, the dry biomass can be subjected to mechanical (e.g., in amill or homogenizer) or chemical (e.g., using an acid, enzyme or base)lysing in the presence of a cold solvent. The cellular debris andprecipitated trisaturated glycerides are separated from the miscella inone step. Post processing steps, such as purification by refining,bleaching and deodorizing, can be performed, if desired.

A second option is to utilize acetone or analogous polar solvent toquantitatively recover lipid from biomass in a conventional extractionprocess (including any type of solvent grinding technique). Thisextraction is conducted at temperatures that solubilize all triglyceridecomponents. Prior to removing cellular debris from the miscella (lipidcontaining triglycerides in solvent), the miscella is chilled toselectively remove the trisaturated glycerides. The chilled miscella isthen centrifuged, filtered, or separated using other techniques toremove both the cellular debris and trisaturated glyceride component.This option combines the concept of extraction and winterization intoone step.

A third option is to utilize acetone or analogous polar solvent toquantitatively recover lipid from biomass in a conventional extractionprocess (including any type of solvent grinding technique). Thisextraction is conducted at temperatures that solubilize all triglyceridecomponents. The cellular debris from the miscella (lipid containingtriglycerides in solvent) is removed using conventional separationtechniques. The miscella is then chilled to crystallize the trisaturatedglycerides, which are removed by centrifugation, filtration, orseparation using other techniques. This option utilizes extraction andwinterization in two stages; however, acetone or an analogous polarsolvent is utilized to accomplish both tasks. A flow diagramillustrating such a process is shown in FIG. 4.

A fourth option is to utilize a nonpolar solvent such as hexane (e.g.,n-hexane, isohexane or a combination thereof) as an extraction solventand utilize acetone as a winterization solvent. Preferably, at least95%, more preferably at least 96%, more preferably at least 97%, morepreferably at least 98% and more preferably at least 99% of the nonpolarsolvent is removed prior to winterization. The winterization step can beemployed at any stage prior to deodorization. A flow diagramillustrating such a process is shown in FIG. 5.

A fifth option is to utilize conventional hexane extraction andhexane-based winterization to remove the majority of the trisaturatedglyceride component and employ a “polishing” step prior to deodorizationto remove the small amounts of trisaturated glycerides contributing tothe haze formation in the lipid. The polishing step employs acetoneand/or an analogous solvent. This option removes the problems caused byhaze, but the lipid level is also reduced.

Preferably, the lipid composition initially comprises at least oneLCPUFA and at least one trisaturated glyceride. Preferably, the other orundesired compound results in the formation of haze when present in theinitial concentration in the initial lipid composition. Preferably, theLCPUFA-containing biomaterial for lipid extraction is selected from thegroup including: LCPUFA-containing microbial biomass or oilseeds fromplants that have been genetically modified to produce LCPUFA containinglipids, particularly plants that have been modified with theLCPUFA-producing genes from microbes (algae, fungi, protists, orbacteria). More preferably, the LCPUFA-containing biomaterial for lipidextraction is selected from the group including thraustochytrid biomass,dinoflagellate biomass and/or Mortierella biomass, and/or oilseeds fromgenetically modified plants containing genes from thraustochytrids,dinoflagellate and/or Mortierella. More preferably, theLCPUFA-containing biomaterial for lipid extraction is selected from thegroup including Schizochytrium, Thraustochytrium and/or Crypthecodinium(preferably, Crypthecodinium cohnii) biomass or oilseeds fromgenetically modified plants containing genes from Schizochytrium orThraustochytrium and/or Crypthecodinium (preferably, Crypthecodiniumcohnii).

Preferably, the initial lipid composition is predominantly made up ofneutral lipids. Preferably, the initial lipid composition comprises atleast 50% neutral lipids, preferably, at least 60% neutral lipids,preferably, at least 75% neutral lipids, preferably at least 85% neutrallipids and preferably at least 90% neutral lipids. Preferably, theneutral lipid predominantly comprises triglyceride. Preferably, theinitial lipid composition comprises at least 50% triglyceride,preferably, at least 60% triglyceride, preferably, at least 75%triglyceride and preferably at least 85% triglyceride. The foregoingpercentages in this paragraph refer to weight percentages. Preferably,the concentration of the desired LCPUFA is greater in the resultingproduct than in the initial lipid composition.

Preferred polar solvent:lipid ratios, based on weight, for theextraction or winterization process are from about 1:10 to about 20:1;more preferably from about 1:8 to about 10:1, preferably from about 1:5to about 5:1, and preferably from about 1:2 to about 2.5:1. Preferablythe contact time between the polar solvent and lipid is from about 0.5to about 12 hours, preferably from about 2 to about 6 hours, andpreferably about 4 hours. Preferably, if a nonpolar lipid is used, theresidual nonpolar lipid is from about 0 to about 4 weight percent, andpreferably from about 1 to about 4 weight percent.

Preferably the temperature for the: (i) cold extraction process, (ii)extraction followed by chilling and filtration/centrifugation, (iii)extraction, filtration/centrifugation of cellular debris, followed bychilling and filtration/centrifugation; and (iv) chilling conditions forsolvent winterization or polishing steps is from the solidificationpoint of the lipid to the melting point of the undesirable component(e.g. trisaturated glycerides), more preferably from about −20° C. toabout 50° C., more preferably from about −5 ° C. to about 20° C., morepreferably from about −5° C. to about 5° C., more preferably about 0° C.

Other preferred attributes of the process include the selective recoveryof only LCPUFA-containing triglycerides at the expense of trisaturatedglycerides and other components that are relatively insoluble in coldacetone including phosphatides, wax esters, saturated fatty acidcontaining sterol esters, sterols, squalene, hydrocarbons and the like.By selectively recovering only the LCPUFA-containing triglyceride at theexpense of these undesirable components allow the possibility ofeliminating or reducing additional downstream purification steps (suchas winterization, refining, and bleaching).

EXAMPLE 1 Summary

A sample of DHA-rich lipid obtained from Schizochytrium (Sample 1,unwinterized lipid, a.k.a. “high melt”) and an isolated sediment fromanother DHA-rich lipid obtained from Schizochytrium (Sample 2) wereanalyzed to determine the nature of the solid phase (Sample 1) and thefloc/sediment (Sample 2).

Unwinterized lipid Sample 1 produced at plant scale (a semi-solid atambient temperature) was dissolved in 4 volumes of cold acetone andmixed. A solid white powder (approximately 7% by weight) was isolated byfiltration through a glass fiber filter. The solid white powder had amelting temperature of 52.4-53.5° C., was shown to be triglycerides(based on a single spot by thin layer chromatography (TLC)), andcontained predominantly myristic (26%) and palmitic acids (66%) whenanalyzed by GLC. This high melting triglyceride fraction containssaturated fatty acids with very little DHA/DPA. The isolated lipidfraction (91% by weight) was an orange-colored liquid at roomtemperature and contained 41.0% DHA and 16.0% DPA. DHA and DPA wereenriched by approximately 8% compared to the starting fatty acid profileof Sample 1—this is a true “purification” of DHA and DPA.

Another DHA-rich reprocessed lipid from Schizochytrium contained anobvious floc-like material (haze) when stored for a period of days atambient temperature. The floc was isolated by centrifugation. Thefloc/sediment (“Sample 2 sediment”) was dissolved in 10 volumes of coldacetone, mixed and filtered. Approximately 15% by weight of a solidwhite powder was isolated by filtration through a glass fiber filter.The solid white powder had a melting temperature of 50.1-51.4° C. andwas shown to be triglycerides (based on single spot by TLC) containingpredominantly myristic (29%) and palmitic acids (59%). This is a highmelting triglyceride fraction containing saturated fatty acids withlittle DHA/DPA. The isolated lipid fraction (85% by weight) was a clear,orange-colored liquid at room temperature and contained 41.1% DHA and16.3% DPA. The floc formation in reprocessed lipid from Schizochytriumis believed to result from a high melting triglyceride, containingmyristic and palmitic fatty acids, which crystallizes from lipid uponstanding.

Experimental

General—A sample of DHA-rich lipid from Sample 1 (250 g bottle) waspulled from frozen storage. This is a sample of unwinterized lipid. Thesample was allowed to warm to ambient temperature and used as is.

Sediment (Sample 2) was isolated from DHA-rich lipid using a labcentrifuge. The DHA-rich lipid was a reprocessed lot of lipid thatcontained a visible floc when left to stand at ambient temperature. Thefloc was isolated by centrifuging the sample and decanting the liquidfraction from the sediment. The liquid fraction remained clear atambient temperature; therefore the floc was believed to be present inthe isolated sediment.

Acetone Winterization—Unwinterized lipid (Sample 1) and sedimentisolated from reprocessed lipid (Sample 2) were fractionated using anacetone winterization procedure. The sediment and unwinterized samplewere dissolved in excess cold acetone (ice/water bath temperature) andmixed to dissolve and suspend lipid components. The solution/suspensionwas immediately filtered through a glass fiber filter under vacuum. Thefilter paper and the contents remaining on the paper were washed withsmall amounts of cold acetone. The contents of the filter paper were airdried and weighed. The lipid/acetone fraction was concentrated undervacuum to afford neat lipid and weighed.

TLC—TLC was performed to determine lipid class composition using silicagel 60 plates. The developing solvent system consisted of a 90:10:1mixture of petroleum ether: ethyl ether: acetic acid. The R_(f) of thespots were compared to those listed in “Techniques in Lipidology” byMorris Kates.

Melting point determination—Melting points were determined using a labconstructed melting point apparatus.

Infrared spectrometry—Infrared spectra were obtained using a PerkinElmer 283B Infrared Spectrometer. Liquid fractions were analyzed neat.Solid fractions from acetone winterization were analyzed in chloroform.

Fatty Acid Methyl Esters (FAMEs)—Aliquots of DHA-rich lipid Sample 1,Sample 2 (reprocessed) along with acetone winterization fractions weretransesterified using anhydrous HCl in methanol following procedures fordetermining the free fatty acid profile, from C12 to C22:6. All FAMEpreparation and GLC work were completed. FAME's were identified andquantified using NuChek Prep analytical reference standard 502 using aninternal standard (C19:0) to determine empirical response factors.

Gas-liquid chromatography—Gas-liquid chromatography of methyl esters wasperformed using a Hewlett-Packard Model 6890 Series II gas-liquidchromatograph equipped with a Hewlett-Packard autosampler, ChemStationsoftware, a 30 m×0.32 mm SP-2380 capillary column (Supelco), and aflame-ionization detector. The oven temperature was held at 120° C. for3 min, programmed to 190° C. at 5° C./min, held at 190° C. for 1 min,programmed to 260° C. at 20° C./min, and then held for 3 minutes at 260°C. The injector temperature was set at 295° C. and the detectortemperature was set at 280° C. Helium was used as a carrier gas and asplit injection technique was employed.

Results DHA-Rich Lipid Sample 1

A sample of unwinterized DHA-rich lipid (250 g bottle) was pulled fromfrozen storage, Sample 1. This sample remained semi-solid at ambienttemperature and can be technically referred to as a “fat”, not an “oil”.An aliquot (14.44 g) of the fat was transferred to an Erlenmeyer flaskand 60 ml of cold acetone (ice/water bath) was added. The flask wasswirled to dissolve/suspend the fat components and immediately filteredthrough a glass fiber filter under vacuum. A solid white fractionremained on the filter paper and was washed with a few milliliters ofcold acetone and dried. The solid white fraction was isolated in a 6.3%yield (0.91 g starting from 14.44 g fat).

The lipid/acetone fraction resulting from filtration was concentrated byrotary evaporation to afford 13.13 g of an orange-colored liquidmaterial (liquid at ambient temperature). This resulted in a 91% overallrecovery; therefore approximately 2% of material was lost at benchscale.

The solid white fraction and the lipid fraction isolated after “acetonewinterization” were analyzed by TLC to determine lipid composition. Thesolid white fraction was shown to be triglycerides based on TLC (onespot with an R_(f) corresponding to a triglyceride was observed). Manyspots were observed by TLC upon spotting and developing the lipidfraction. The R_(f) of the spots was consistent with lipid componentscomprising squalene, steryl esters, triglycerides, and sterols (alltentative assignments). No further analysis of lipid class compositionwas performed.

The solid white fraction isolated after acetone winterization had amelting point range of 52.4-53.5° C.

The solid and liquid fraction isolated after acetone winterization weretransesterified to methyl esters and the methyl esters were analyzed bygas-liquid chromatography. The complete profile of FAME's for both thesolid and liquid fraction isolated by acetone winterization along withunwinterized DHA-rich fat (Sample 1) is shown in Table 1. As is evident,the solid fraction contained very little DHA (2.4%) and DPA (0.9%) withmethyl myristate (26%) and methyl palmitate (66%) as the predominantfatty acids. The liquid fraction isolated after acetone winterizationcontained myristate (8.3%), palmitate (23.1%), DPA (16.0%), DHA (41.0%)along with other minor fatty acids. When this profile is compared tothat of the starting unwinterized lipid, an enrichment of the DHA ofapproximately 8% is seen, consistent with the removal of thepredominantly trisaturated glyceride component. This represents apurification step.

DHA-Rich Lipid Sediment (Sample 2)

The sediment that was produced from re-refined lipid was completelymiscible in hexane and not miscible in methanol. When small quantitiesof acetone were added to the sediment, a white precipitate formed whichseparated from the liquid, yellow-colored lipid/acetone phase. Based onthese dissolution tests, acetone fractionation was used to isolate thewhite powder.

An aliquot (1.11 g) of sediment was transferred to an Erlenmeyer flaskand 10 ml of cold acetone (ice/water bath) was added. The flask wasswirled to dissolve/suspend the fat components and immediately filteredthrough a glass fiber filter under vacuum. A solid white fractionremained on the filter paper and was washed with a few milliliters ofcold acetone and dried. The solid white fraction was isolated in a 15%yield (0.17 g starting from 1.11 g sediment).

The lipid/acetone fraction resulting from filtration was concentrated byrotary evaporation to afford 0.94 g of an orange-colored liquid material(liquid at ambient temperature). This resulted in an 85% overallrecovery.

The solid white fraction and the lipid fraction isolated after acetonefractionation were analyzed by TLC to determine lipid composition. Thesolid white fraction was shown to be triglycerides based on TLC (onespot with an R_(f) corresponding to a triglyceride was observed). Manyspots were observed by TLC upon spotting and developing the lipidfraction. The R_(f) of the spots was consistent with lipid componentscomprising squalene, steryl esters, triglycerides, and sterols (alltentative assignments). No further analysis of lipid class compositionwas performed.

The solid white fraction isolated after acetone winterization had amelting point range of 50.1-51.4° C.

The solid and liquid fraction isolated after acetone winterization weretransesterified to methyl esters and the methyl esters were analyzed bygas-liquid chromatography. The complete profile of FAME's for both thesolid and liquid fraction isolated by acetone winterization along withSample 2 sediment is shown in Table 1. As is evident, the solid fractioncontains very little DHA (6.4%) and DPA (2.6%) with methyl myristate(29%) and methyl palmitate (59%) as the predominant fatty acids. Theliquid fraction isolated after acetone winterization contains myristate(8.4%), palmitate (23.2%), DPA (16.3%), DHA (41.1%) along with otherminor fatty acids.

TABLE 1 Fatty acid profile of unwinterized oil (Sample 1), Sample 2sediment and fractions isolated from Sample 1 and Sample 2 sediment byacetone fractionation Isolated Isolated Isolated Solid Liquid IsolatedLiquid Fraction Fraction Lot Unwinterized Solid Fraction Sample 2 Sample2 21A FA Name Sample 1 Fraction Sample 1 Sediment Sediment Sediment 14:09.6 25.9 8.3 12.2 27.0 8.4 16:0 25.9 66.0 23.1 30.5 58.8 23.2 16:1 0.3<0.1 0.3 0.3 0.2 0.3 18:0 0.7 1.8 0.6 0.7 1.5 0.6 18:4 n3 0.4 <0.1 0.40.3 <0.1 0.4 20:3 n6 0.4 0.2 0.4 0.3 0.2 0.5 20:4 n7 2.8 <0.1 2.6 1.8<0.1 2.4 20:4 n6 0.9 <0.1 1.0 0.8 0.1 1.0 20:4 n3 0.8 <0.1 0.9 0.8 <0.10.9 20:5 n3 2.2 <0.1 2.3 1.9 0.3 2.3 22:4 n9 0.2 <0.1 0.1 0.2 <0.1 0.222:5 n6 14.7 0.9 16.0 13.6 2.6 16.3 22:6 n3 37.7 2.4 41.0 34.2 6.4 41.1

COMPARATIVE EXAMPLE

Table 2, set forth below, represents a comparative prior method as shownin Comparative FIG. 1 followed by Comparative FIG. 2.

TABLE 2 Certificate of Analysis (Schizochytrium Biomass) Refined,Deodorized, Bleached (RDB) Winterized Schizochytrium oil afterantioxidants addition Specification Result Method Reference PeroxideValue, Maximum 3.0 0.42 AOCS Cd 8-53 meq/kg Free Fatty Acids, % Maximum0.25 0.06 AOCS Ca 5a-40 Moisture and Maximum 0.05 0.03 AOCS Ca 2d-25volatiles, % Trace Metals, ppm POS AS.SOP-103 Lead Maximum 0.20 <0.20Arsenic Maximum 0.20 <0.20 Iron Maximum 0.20 0.04 Copper Maximum 0.05<0.05 Mercury Maximum 0.20 <0.20 DHA, % of FAME, Minimum 32.0 43.5 POSAS.SOP-104 wt/wt DHA, mg/g of oil Minimum 300 397.3 POS AS.SOP-104Residual Hexane, Maximum 10 <1.0 AOCS Ca 3b-87 ppm Specification ValueMethod Reference Neutral oil, % N/A 99.69 p-Anisidine Value N/A 0.74AOCS Cd 18-90 Colour, 1.0″ Lovibond N/A 70.0Y AutoTintometer (PFX 990AOCS) 7.1R Colour Colour, Gardner Scale, N/A 12.3 (1 cm) β- Carotene(PFX990), N/A 276.41 ppm, (0.01 cm) Note: not true β- CaroteneUnsaponifiables, % N/A 2.24 AOCS Ca 6b-53 Insoluble Impurities, % N/A0.01 AOCS Ca 3-46 AOM, hr N/A 7.66 AOCS Cd 12-57 Rancimat (80° C.), HrN/A 22.7 Spin test, % solids by N/A ~0.2* volume, 20° C./24 hrs afterantiox addition Spin test, % solids by N/A zero Vol, before antioxaddition Fatty Acid Composition N/A POS AS.SOP-104 (absolute), mg/g C122.6 C14 69.4 C14:1 0.8 C15 3.1 C16 187.8 C16:1 4.4 C18 4.6 C18:1 7.2C18:2 3.6 C18:3n6 2.3 C18:4 3.0 C20 1.2 C20:4n6 7.4 C20:4n3 AA 8.5C20:5n3 EPA 18.2 C22 0.6 C22:5n6† DPA 151.6 C22:6n3 DHA 397.3 C24 1.8C24:1 1.9 Others 35.1 Total, mg/g 912.4 DHA, % of FAME 43.5 Ascorbylpalmitate, ppm 224 Tocopherols, ppm 1,760 *ppte from Addition ofRosemary extract.

Table 3, set forth below, represents a process of the present invention,as set forth in FIG. 5 followed by the bleaching, deodorizing andrefining of Comparative FIG. 2.

TABLE 3 Acetone Winterized Schizo oil RDB Schizo oil after antioxidantsaddition (From Schizochytrium biomass) Specification Result MethodReference Peroxide Value, Maximum 3.0 1.32 AOCS Cd 8-53 meq/kg FreeFatty Acids, % Maximum 0.25 0.06 AOCS Ca 5a-40 Moisture and Maximum 0.050.03 AOCS Ca 2d-25 volatiles, % Trace Metals, ppm POS AS.SOP-103 LeadMaximum 0.20 <0.20 Arsenic Maximum 0.20 <0.20 Iron Maximum 0.20 0.11Copper Maximum 0.05 <0.05 Mercury Maximum 0.20 <0.20 DHA, % of FAMEMinimum 32.0 42.8 POS AS.SOP-104 DHA, mg/g of oil Minimum 300 385.5 POSAS.SOP-104 Residual Hexane, Maximum 10 <1.0 AOCS Ca 3b-87 ppmSpecification Value Method Reference Neutral oil, % N/A 99.69p-Anisidine Value N/A 1.08 AOCS Cd 18-90 Colour, 1.0″ Lovibond N/A 70.0YAutoTintometer (PFX 990 AOCS) 6.3R Colour Colour, Gardner Scale, N/A12.0 (1 cm) β- Carotene (PFX990), N/A 228.0 ppm, (0.01 cm) Note: nottrue β- Carotene Unsaponifiables, % N/A 2.11 AOCS Ca 6b-53 InsolubleImpurities, % N/A 0.01 AOCS Ca 3-46 AOM, hr N/A 7.00 AOCS Cd 12-57Rancimat (80° C.), Hr N/A 19.9 Spin test, % solids by N/A ≈0.2 volume,20° C./24 hrs Fatty Acid Composition N/A POS AS.SOP-104 (absolute), mg/gC12 3.9 C14 90.1 C14:1 0.8 C15 3.4 C16 193.9 C16:1 6.5 C18 4.8 C18:1 8.1C18:2 3.6 C18:3n6 1.7 C18:4 2.6 C20 1.5 C20:4n6 4.9 C20:4n3 AA 7.7C20:5n3 EPA 12.5 C22 0.8 C22:5n6† DPA 129.7 C22:6n3 DHA 385.5 C24 1.9C24:1 1.6 Others 34.5 Total, mg/g 900.0 DHA, % of FAME 42.8 Ascorbylpalmitate, ppm 222 Tocopherols, ppm 1940

EXAMPLE 3

A crude extract of Schizochytrium oil was subjected to a variety ofwinterization procedures in which a lipid composition was extracted frombiomass with hexane. The hexane was removed to produce a crude extractedoil having a residual amount of hexane. The extracted oil was thenextracted with acetone at a particular acetone/oil ratio and winterizedat a particular temperature for a given amount of time. The % residualhexane, acetone/oil ratio, winterization temperature and winterizationtime were varied in different experiments. The processes were evaluatedin terms of filtration time, oil recovery and haziness after two weeks.The details of the experiments and the results are shown below in Table4.

TABLE 4 The levels of tested variables and observations ofacetone-winterized Schizochytrium oil Winter- Winter- Oil HazinessExperiment Hexane Acetone/ ization ization Filtration Recovery After 2No. % Oil Ratio Temp. (C.) Time (H) @ (sec) (%) weeks 1 1 1.5 5 3 6787.8 Clear 2 2 1 0 2 165 86.4 PPT 3 2 1 0 4 195 87.7 Clear 4 2 1 10 2178 88.1 PPT 5 2 1 10 4 154 89.8 PPT 6 2 2 0 2 85 84.1 PPT 7 2 2 0 4 7586.2 Clear 8 2 2 10 2 67 88.9 PPT 9 2 2 10 4 82 86.7 PPT 10 3 0.5 5 3264 84.3 PPT 11 3 1.5 −5 3 102 83.4 Clear 12 3 1.5 5 1 87 85.5 PPT 13 31.5 5 3 109 85.4 Clear 14 3 1.5 5 3 123 86.3 Clear 15 3 1.5 5 3 82 87.5Clear 16 3 1.5 5 3 110 87.9 Clear 17 3 1.5 5 5 117 86.6 PPT 18 3 1.5 153 255 94.8 PPT 19 3 2.5 5 3 73 87.2 PPT 20 4 1 0 2 262 87.5 Clear 21 4 10 4 115 91.2 PPT 22 4 1 10 2 245 83.7 PPT 23 4 1 10 4 375 86.7 PPT 24 42 0 2 52 88.4 PPT 25 4 2 0 4 80 89.3 PPT 26 4 2 10 2 92 86.8 PPT 27 4 210 4 83 88.7 PPT 28 5 1.5 5 3 86 87.1 PPT Control 150 90.9  PPT*Control: Hexane winterization (45:55, Oil:Hexane) at −3 C. for 5 hPPT—Precipitate observed after spin-test *The hexane winterized sampleshowed PPT after filtration (the same day), an indication of incompletecrystallization. The recovery obtained in the lab would not beduplicated in the plant as the thorough drying of the cake may not beachievable with the enclosed filters. Typical recovery in plant isaround 70-75%.

TABLE 5 The oil recovery, filtration time and analytical data of crudeoil, hexane and acetone-winterized oils. Plant- Lab- Acetone- Acetone-Hexane Hexane- winterized oil winterized oil Observations/ winterizedwinterized (Verification (Verification analysis Crude oil oil oiltrail-1) trail-2) Oil recovery (%) 70% 90.9 86.9 85.3 Filtration @ (Sec)— 150 158 114 Color (1″ cell) Too dark — 70Y Too dark Too dark (1 cmcell) 70Y 11.2R 12.3R 70Y 12R 70Y 11.1R Phosphorus (ppm) 474.3 — 474.0271.6 144.3 Free fatty acids 0.53 — 0.49 0.52 0.43 PV (meq/kg) 0.00 —1.82 3.32 4.27 Anisidine value 4.11 — 4.37 3.73 3.66 Fatty acid comp.(mg/g) C12:0 2.3 — 2.1 2.2 2.1 C14:0 67.2 — 57.8 58.5 58.9 C14:1 0.7 —0.7 0.8 0.8 C15:0 3.3 — 3.1 3.1 3.2 C16:0 204.9 — 185.2 187.0 188.1C16:1 3.3 — 3.5 3.6 3.5 C18:0 5.1 — 4.5 4.5 4.7 C18:1 3.9 — 4.0 4.0 4.0C18:2 2.6 — 2.7 2.7 2.7 C18:3n6 2.3 — 2.5 2.5 2.6 C18:4 3.3 — 3.5 3.63.6 C20:0 1.2 — 1.0 1.0 1.0 C20:4n6 9.4 — 9.7 10.2 10.3 C20:4n3 8.0 —8.3 8.5 8.6 C20:5n3 23.6 — 24.9 25.4 25.6 C22:0 0.6 — 0.6 0.5 0.6C22:5n6 142.9 — 149.6 152.7 154.0 C22:6n3 351.1 369.0* 369.0 378.6 382.2C24:0 1.9 — 1.6 1.6 1.6 C24:1 4.0 — 4.1 4.3 4.2 Others 35.2 — 37.5 37.938.4 Recovery of DHA 73% 95.6% 93.8% 92.8% *The estimation of DHArecovery of Pilot Plant hexane -winterized oil is based on the past dataof Schizo oil process

TABLE 6 The fatty acid composition of acetone-winterized wax.Observations/ Acetone-winterized wax Acetone-winterized wax Analysis(Verification trail-1) (Verification trail-2) Wax recovery (%) 13.1 14.7Fatty acid comp. (mg/g) C12:0 2.8 2.6 C14:0 112.4 103.2 C14:1 0.4 0.4C15:0 3.8 0.6 C16:0 303.4 282.6 C16:1 2.1 2.1 C18:0 8.6 8.7 C18:1 3.33.6 C18:2 1.3 1.7 C18:3n6 1.1 1.0 C18:4 1.5 1.4 C20:0 2.2 2.0 C20:4n64.9 4.6 C20:4n3 4.1 4.0 C20:5n3 11.9 11.9 C22:0 1.3 1.2 C22:5n6 76.875.7 C22:6n3 175.2 170.5 C24:0 3.8 3.5 C24:1 2.1 2.0 Others 16.6 18.1

CONCLUSIONS

Based on an analysis of the Sample 2 sediment, it is believed the flocis triglycerides containing predominantly myristic and palmitic acids.This is based on TLC, IR, and resulting FAME analysis by GLC. Thetriglycerides comprising the floc had a high melting temperature(50.1-51.4° C.).

The high melting temperature of the isolated white powder, coupled withthe triglyceride lipid class composition of this fraction, indicatesthat the winterization step employed during standard processing is notquantitatively removing “high melting” fractions from the lipid.Therefore, an additional “polishing” step is recommended to achieveclarity in the finished goods product.

To estimate the solid contribution of unwinterized lipid in Sample 1, anacetone winterization procedure was employed. A solid white fractionisolated from Sample 1 in 6-7% yield was shown to be triglyceridescontaining predominantly myristic and palmitic acids (>94% of the fattyacids in this triglyceride component were saturated fats). Palmitic andmyristic acid are present in roughly a 2:1 ratio and, coupled with thenarrow range in melting temperature, suggest a defined structure to thistriglyceride. Very little DPA and DHA were present in the solidtriglyceride fraction. The isolated liquid fraction following acetonewinterization contained 41.0% DHA (expressed as a percentage of totalfatty acid methyl esters) compared to 37.7% DHA in the startingunwinterized lipid. This is an approximate 8% enrichment of DHA,consistent with the removal of 7% trisaturated fatty acid glycerides.

Very little loss of DHA was shown in the bench scale acetonewinterization process, indicating near quantitative recovery of DHA canbe obtained during winterization.

Solid or solvent assisted winterization (acetone winterizationdemonstrated herein, however other solvent alternatives exist) offer thefollowing possibilities and can be considered as processing options.

-   -   (1) A true removal of high melting, solid material can be        accomplished.    -   (2) The solid material is mainly trisaturated fatty acid        glyceride (>94% saturated fatty acids) with very little DHA        (2.4%).    -   (3) As an example calculation, starting from 1,000 kg's of DHA        in crude lipid, an approximate loss of 2 kg's of DHA would be        encountered during acetone winterization (1,000×0.07×0.024).        This is approximately a 0.2% recovery loss of DHA on an absolute        weight basis.    -   (4) A clear liquid remains following winterization, with        enrichment of DHA compared to the starting unwinterized lipid        fatty acid profile.    -   (5) Solvent assisted winterization can be used to achieve DHA        purification.    -   (6) Because of the high melting temperature of the trisaturated        fatty acid glyceride component (>50° C.), traditional low        temperature chilling conditions may not be required.

This application incorporates by reference U.S. Provisional PatentApplication No. 60/341,180, filed on Dec. 12, 2001.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims.

1-47. (canceled)
 48. A process for recovering a lipid compositioncomprising predominantly neutral lipid from a biomass, wherein thebiomass comprises cellular debris, at least one long chainpolyunsaturated fatty acid (LCPUFA) and at least one other compound, theprocess comprising the steps: (a) contacting the biomass with a nonpolarsolvent to recover lipid in an extraction process; (b) processing thelipid composition by a step selected from the group consisting ofrefining the lipid composition, bleaching the lipid composition, anddeodorizing the lipid composition; (c) adding a polar solvent to thelipid composition; (d) cooling the polar solvent and lipid compositionto selectively precipitate at least one other compound; and (e)separating the precipitated other compound from the lipid composition.49. The process of claim 48, wherein the step of processing the lipidcomposition comprises processing the lipid composition by two or moresteps selected from the group consisting of refining the lipidcomposition, bleaching the lipid composition, and deodorizing the lipidcomposition.
 50. The process of claim 48, wherein the step of processingthe lipid composition comprises refining the lipid composition,bleaching the lipid composition and deodorizing the lipid composition.51. The process of claim 48, wherein the step of processing the lipidcomposition is conducted after the step of cooling of the polar solventand lipid composition.
 52. The process of claim 48, wherein the LCPUFAis obtained from a source from the group consisting of Schizochytriumbiomass, Thraustochytrium biomass and Crypthecodinium cohnii biomass andoilseeds from genetically modified plants containing genes fromSchizochytrium or Thraustochytrium.
 53. The process of claim 48, whereinthe LCPUFA is docosahexaenoic acid (DHA).
 54. The process of claim 48,wherein the other compound is selected from the group consisting oftrisaturated glycerides, phosphorus-containing materials, wax esters,saturated fatty acid containing sterol esters, sterols, squalene, andhydrocarbons.
 55. The process of claim 48, wherein the other compound isselected from the group consisting trisaturated glycerides of lauric(C12:0), myristic (C14:0), palmitic (C16:0) and stearic (C18:0) fattyacids and mixtures thereof.
 56. The process of claim 48, wherein theother compound results in the formation of haze when present in theinitial concentration in the initial lipid composition.
 57. The processof claim 48, wherein the nonpolar solvent is hexane.
 58. The process ofclaim 48, wherein most of the nonpolar solvent is removed from the lipidcomposition.
 59. The process of claim 48, wherein the nonpolar solventafter removing most of the nonpolar solvent from the lipid compositionis from about 0 to about 4 weight percent of the lipid composition. 60.The process of claim 48, wherein the nonpolar solvent after removingmost of the nonpolar solvent from the lipid composition is from about 1to about 4 weight percent of the lipid composition.
 61. The process ofclaim 48, wherein the time of contact between the polar solvent and thelipid composition is from about 0.5 to about 12 hours.
 62. The processof claim 48, wherein the time of contact between the polar solvent andthe lipid composition is from about 2 to about 6 hours.
 63. The processof claim 48, wherein the time of contact between the polar solvent andthe lipid composition is about 5 hours.
 64. The process of claim 48,wherein the polar solvent is acetone.
 65. The process of claim 48,wherein step (e) separating the precipitated other compound from thelipid composition is selected from the group consisting ofcentrifugation, filtering and a combination thereof.
 66. The process ofclaim 48, wherein step (e) separating the precipitated other compoundfrom the lipid composition is by filtering.
 67. The process of claim 48,wherein said cooling of the polar solvent and lipid composition toselectively precipitate at least one other compound occurs at atemperature that the desirable LCPUFAs are soluble in the polar solvent,and the undesirable compounds are not soluble in the polar solvent. 68.A process for recovering a lipid composition comprising predominantlyneutral lipid from a biomass, wherein the biomass comprises cellulardebris, at least one long chain polyunsaturated fatty acid (LCPUFA) andat least one other compound, the process comprising the steps: (a)contacting the biomass with a nonpolar solvent to recover lipid in anextraction process; (b) processing the lipid composition by a stepselected from the group consisting of refining the lipid composition,bleaching the lipid composition, and deodorizing the lipid composition;(c) adding a polar solvent to the lipid composition; (d) cooling thepolar solvent and lipid composition to selectively precipitate at leastone other compound; and (e) filtering the precipitated other compoundfrom the lipid composition.